Investigation of CaMV-host co-evolution through synonymous codon pattern.

Cauliflower mosaic virus (CaMV) has a double-stranded DNA genome and is globally distributed. The phylogeny tree of 121 CaMV isolates was categorized into two primary groups, with Iranian isolates showing the greatest genetic variations. Nucleotide A demonstrated the highest percentage (36.95%) in the CaMV genome and the dinucleotide odds ratio analysis revealed that TC dinucleotide (1.34 ≥ 1.23) and CG dinucleotide (0.63 ≤ 0.78) are overrepresented and underrepresented, respectively. Relative synonymous codon usage (RSCU) analysis confirmed codon usage bias in CaMV and its hosts. Brassica oleracea and Brassica rapa, among the susceptible hosts of CaMV, showed a codon adaptation index (CAI) value above 0.8. Additionally, relative codon deoptimization index (RCDI) results exhibited the highest degree of deoptimization in Raphanus sativus. These findings suggest that the genes of CaMV underwent codon adaptation with its hosts. Among the CaMV open reading frames (ORFs), genes that produce reverse transcriptase and virus coat proteins showed the highest CAI value of 0.83. These genes are crucial for the creation of new virion particles. The results confirm that CaMV co-evolved with its host to ensure the optimal expression of its genes in the hosts, allowing for easy infection and effective spread. To detect the force behind codon usage bias, an effective number of codons (ENC)-plot and neutrality plot were conducted. The results indicated that natural selection is the primary factor influencing CaMV codon usage bias.

Comparison of two Turnip mosaic virus P1 proteins in their ability to co-localize with the Arabidopsis thaliana G3BP-2 protein.

Turnip mosaic virus (TuMV), belonging to the genus Potyvirus (family Potyviridae), has a large host range and consists of a single-stranded positive sense RNA genome encoding 12 proteins, including the P1 protease. This protein which is separated from the polyprotein by cis cleavage at its respective C-terminus, has been attributed with different functions during potyviral infection of plants. P1 of Turnip mosaic virus (P1-TuMV) harbors an FGSF-motif and FGSL-motif at its N-terminus. This motif is predicted to be a binding site for the host Ras GTPase-activating protein-binding protein (G3BP), which is a key factor for stress granule (SG) formation in mammalian systems and often targeted by viruses to inhibit SG formation. We therefore hypothesized that P1-TuMV might interact with G3BP to control and regulate plant SGs to optimize cellular conditions for the production of viral proteins. Here, we analyzed the co-localization of the Arabidopsis thaliana G3BP-2 with the P1 of two TuMV isolates, namely UK 1 and DEU 2. Surprisingly, P1-TuMV-DEU 2 co-localized with AtG3BP-2 under abiotic stress conditions, whereas P1-TuMV-UK 1 did not. AtG3BP-2::RFP showed strong SGs formation after stress, while P1-UK 1::eGFP maintained a chloroplastic signal under stress conditions, the signal of P1-DEU 2::eGFP co-localized with that of AtG3BP-2::RFP. This indicates a specific interaction between P1-DEU 2 and the AtG3BP family which is not solely based on the canonical interaction motifs.

The 2b protein and C-terminal region of the 2a protein indispensably facilitate systemic movement of cucumber mosaic virus in radish with supplementary function by either the 3a or the coat protein.

BACKGROUND: In Raphanus sativus (Japanese radish), strain D8 of cucumber mosaic virus (CMV-D8) establishes a systemic infection and induces mild mosaic on upper, non-inoculated leaves, whereas strain Y of CMV (CMV-Y) causes only a local infection in the inoculated leaves. Here, we further analyzed the specific viral factor(s) of CMV-D8 that is (are) indispensable for systemic infection in Japanese radish. METHODS: To identify which genomic RNA(s) is (are) involved in systemic infection in radish, we carried out a pseudorecombination analysis between CMV-D8 and CMV-Y. With recombination analyses between CMV-D8 and CMV-Y using mutant/recombinant RNA2s, chimeric and point-mutated RNA3s, we identified viral factors that are indispensable for systemic infection. RESULTS: Viral RNA2 and RNA3 of CMV-D8 facilitated efficient virus spread into the upper, non-inoculated plant tissues of radish (cv. Tokinashi), but not those of CMV-Y. Recombinant RNA2s demonstrated that the 2b protein (2b) and the C-terminus of the 2a protein (2a) of CMV-D8 have a crucial role in systemic infection. In addition, we used chimeric and point-mutated RNA3s to that Pro17 and Pro129 in the coat protein (CP) of CMV-D8 are involved in efficient systemic infection and that Ser51 in the 3a protein (3a) of CMV-D8 has positive effects on systemic spread. The results suggested that these viral factors facilitate systemic infection of CMV-D8 in Japanese radish. CONCLUSION: The C-terminal region of 2a, the entire region of 2b, and supplementary function of either Ser51 in 3a or Pro17/Pro 129 in CP confer systemic infectivity on CMV-D8 in radish. These results further elucidate the complex interaction of viral proteins of CMV to complete systemic infection as a host-specific manner.

Molecular characterization of a new recombinant brassica yellows virus infecting tobacco in China.

Brassica yellows virus (BrYV), prevalently distributed throughout mainland China and South Korea while triggering serious diseases in cruciferous crops, is proposed to be a new species in the genus Polerovirus within the family Luteoviridae. There are three distinct genotypes (BrYV-A, BrYV-B and BrYV-C) reported in cabbage and radish. Here, we describe a new BrYV isolate infecting tobacco plants in the field, which was named BrYV-NtabQJ. The complete genome sequence of BrYV-NtabQJ is 5741 nt in length, and 89% of the sequence shares higher sequence identities (about 90%) with different BrYV isolates. However, it possesses a quite divergent region within ORF5, which is more close to Beet western yellows virus (BWYV), Beet mild yellowing virus (BMYV) and Beet chlorosis virus (BChV). A significant recombination event was then detected among BrYV-NtabQJ, BrYV-B Beijng isolate (BrYV-BBJ) and BWYV Leonurus sibiricus isolate (BWYV-LS). It is proposed that BrYV-NtabQJ might be an interspecific recombinant between BrYV-BBJ and BWYV-LS, and the recombination might result in the successful aphid transmission of BrYV from cruciferous crops to tobacco. And it also poses new challenges for BrYV diagnosis and the vegetable production.

Sequence Variations Among 17 New Radish Isolates of Turnip mosaic virus Showing Differential Pathogenicity and Infectivity in Nicotiana benthamiana, Brassica rapa, and Raphanus sativus.

Infectious clones were generated from 17 new Korean radish isolates of Turnip mosaic virus (TuMV). Phylogenetic analysis indicated that all new isolates, and three previously characterized Korean radish isolates, belong to the basal-BR group (indicating that the pathotype can infect both Brassica and Raphanus spp.). Pairwise analysis revealed genomic nucleotide and polyprotein amino acid identities of >87.9 and >95.7%, respectively. Five clones (HJY1, HJY2, KIH2, BE, and prior isolate R007) had lower sequence identities than other isolates and produced mild symptoms in Nicotiana benthamiana. These isolates formed three distinct sequence classes (HJY1/HJY2/R007, KIH2, and BE), and several differential amino acid residues (in P1, P3, 6K2, and VPg) were present only in mild isolates HJY1, HJY2, and R007. The remaining isolates all induced systemic necrosis in N. benthamiana. Four mild isolates formed a phylogenetic subclade separate from another subclade including all of the necrosis-inducing isolates plus mild isolate KIH2. Symptom severity in radish and Chinese cabbage genotypes was not correlated with pathogenicity in N. benthamiana; indeed, Chinese cabbage cultivar Norang was not infected by any isolate, whereas Chinese cabbage cultivar Chusarang was uniformly susceptible. Four isolates were unable to infect radish cultivar Iljin, but no specific amino acid residues were correlated with avirulence. These results may lead to the identification of new resistance genes against TuMV.

Complete genomic sequence of Raphanus sativus cryptic virus 4 (RsCV4), a novel alphapartitivirus from radish.

The present work reports the discovery and complete genome sequencing of a virus from symptomless radish seedlings, classifiable as a novel member of the genus Alphapartitivirus, family Partitiviridae. Total RNA extracted from germinating seedlings was sequenced using Illumina technology. Bioinformatic analysis of the RNA-seq data revealed two contigs representing the near full-length genomic sequences of two genomic RNAs representing a new virus. Analysis of the genome sequence (excluding the polyA tail, RNA1: 1976 nt and RNA2: 1751 nt, respectively) showed a genomic organization typical of viruses classed within the Partitiviridae, with each genomic RNA encoding a single open reading frame (ORF). Phylogenetic analysis of the RNA dependent RNA polymerase (RNA1 ORF) and of the capsid protein (RNA2 ORF) clearly showed the new virus can be classified within the genus Alphapartitivirus, but sequence divergence establishes it as a new species, for which the name "Raphanus sativus cryptic virus 4" is proposed.

Internalization and dissemination of human norovirus and Tulane virus in fresh produce is plant dependent.

Human norovirus (NoV) is a leading cause of fresh produce associated outbreaks. Previous research indicates that the roots of growing leafy greens and berries internalize human NoV. However the effect of plant type and inoculum level on internalization rates has not been directly compared. In this study we compared the internalization and dissemination rates of human NoV and its surrogate, Tulane virus (TV) in green onion, radishes, and Romaine lettuce. We also evaluated the effect inoculum level and plant growth matrix on the rate of viral internalization. In the hydroponic growth system, we detected internalization and dissemination of human NoV RNA in green onions. In hydroponically growing green onions inoculated with high titer TV, we found higher rates of internalization and dissemination compared to green onions inoculated with low titer TV. In soil growth systems, no infectious TV was detected in either green onion or radishes. However, in Romaine lettuce plants grown in soil approximately 4 log10 PFU/g was recovered from all tissues on day 14 p.i. Overall, we found that the type of plant, growth matrix, and the inoculum level influences the internalization and dissemination of human NoV and TV.

The genetic structure of Turnip mosaic virus population reveals the rapid expansion of a new emergent lineage in China.

BACKGROUND: Turnip mosaic virus (TuMV) is one of the most widespread and economically important virus infecting both crop and ornamental species of the family Brassicaceae. TuMV isolates can be classified to five phylogenetic lineages, basal-B, basal-BR, Asian-BR, world-B and Orchis. RESULTS: To understand the genetic structure of TuMV from radish in China, the 3'-terminal genome of 90 TuMV isolates were determined and analyzed with other available Chinese isolates. The results showed that the Chinese TuMV isolates from radish formed three groups: Asian-BR, basal-BR and world-B. More than half of these isolates (52.54%) were clustered to basal-BR group, and could be further divided into three sub-groups. The TuMV basal-BR isolates in the sub-groups I and II were genetically homologous with Japanese ones, while those in sub-group III formed a distinct lineage. Sub-populations of TuMV basal-BR II and III were new emergent and in a state of expansion. The Chinese TuMV radish populations were under negative selection. Gene flow between TuMV populations from Tai'an, Weifang and Changchun was frequent. CONCLUSIONS: The genetic structure of Turnip mosaic virus population reveals the rapid expansion of a new emergent lineage in China.

Molecular Characterization of the Complete Genome of Three Basal-BR Isolates of Turnip mosaic virus Infecting Raphanus sativus in China.

Turnip mosaic virus (TuMV) infects crops of plant species in the family Brassicaceae worldwide. TuMV isolates were clustered to five lineages corresponding to basal-B, basal-BR, Asian-BR, world-B and OMs. Here, we determined the complete genome sequences of three TuMV basal-BR isolates infecting radish from Shandong and Jilin Provinces in China. Their genomes were all composed of 9833 nucleotides, excluding the 3'-terminal poly(A) tail. They contained two open reading frames (ORFs), with the large one encoding a polyprotein of 3164 amino acids and the small overlapping ORF encoding a PIPO protein of 61 amino acids, which contained the typically conserved motifs found in members of the genus Potyvirus. In pairwise comparison with 30 other TuMV genome sequences, these three isolates shared their highest identities with isolates from Eurasian countries (Germany, Italy, Turkey and China). Recombination analysis showed that the three isolates in this study had no "clear" recombination. The analyses of conserved amino acids changed between groups showed that the codons in the TuMV out group (OGp) and OMs group were the same at three codon sites (852, 1006, 1548), and the other TuMV groups (basal-B, basal-BR, Asian-BR, world-B) were different. This pattern suggests that the codon in the OMs progenitor did not change but that in the other TuMV groups the progenitor sequence did change at divergence. Genetic diversity analyses indicate that the PIPO gene was under the highest selection pressure and the selection pressure on P3N-PIPO and P3 was almost the same. It suggests that most of the selection pressure on P3 was probably imposed through P3N-PIPO.

Seed-borne viral dsRNA elements in three cultivated Raphanus and Brassica plants suggest three cryptoviruses.

Since the 1970s, several dsRNA viruses, including Radish yellow edge virus, Raphanus sativus virus 1, Raphanus sativus virus 2, and Raphanus sativus virus 3, have been identified and reported as infecting radish. In the present study, in conjunction with a survey of seed-borne viruses in cultivated Brassica and Raphanus using the dsRNA diagnostic method, we discovered 3 novel cryptoviruses that infect Brassica and Raphanus: Raphanus sativus partitivirus 1, which infects radish (Raphanus sativus); Sinapis alba cryptic virus 1, which infects Sinapis alba; and Brassica rapa cryptic virus 1 (BrCV1), which infects Brassica rapa. The genomic organization of these cryptoviruses was analyzed and characterized. BrCV1 might represent the first plant partitivirus found in Gammapartitivirus. Additionally, the evolutionary relationships among all of the partitiviruses reported in Raphanus and Brassica were analyzed.

Cell Death Triggered by a Putative Amphipathic Helix of Radish mosaic virus Helicase Protein Is Tightly Correlated With Host Membrane Modification.

Systemic necrosis is one of the most severe symptoms caused by plant RNA viruses. Recently, systemic necrosis has been suggested to have similar features to a defense response referred to as the hypersensitive response (HR), a form of programmed cell death. In virus-infected plant cells, host intracellular membrane structures are changed dramatically for more efficient viral replication. However, little is known about whether this replication-associated membrane modification is the cause of the symptoms. In this study, we identified an amino-terminal amphipathic helix of the helicase encoded by Radish mosaic virus (RaMV) (genus Comovirus) as an elicitor of cell death in RaMV-infected plants. Cell death caused by the amphipathic helix had features similar to HR, such as SGT1-dependence. Mutational analyses and inhibitor assays using cerulenin demonstrated that the amphipathic helix-induced cell death was tightly correlated with dramatic alterations in endoplasmic reticulum (ER) membrane structures. Furthermore, the cell death-inducing activity of the amphipathic helix was conserved in Cowpea mosaic virus (genus Comovirus) and Tobacco ringspot virus (genus Nepovirus), both of which are classified in the family Secoviridae. Together, these results indicate that ER membrane modification associated with viral intracellular replication may be recognized to prime defense responses against plant viruses.

Complete genome sequence analysis identifies a new genotype of brassica yellows virus that infects cabbage and radish in China.

For brassica yellows virus (BrYV), proposed to be a member of a new polerovirus species, two clearly distinct genotypes (BrYV-A and BrYV-B) have been described. In this study, the complete nucleotide sequences of two BrYV isolates from radish and Chinese cabbage were determined. Sequence analysis suggested that these isolates represent a new genotype, referred to here as BrYV-C. The full-length sequences of the two BrYV-C isolates shared 93.4-94.8 % identity with BrYV-A and BrYV-B. Further phylogenetic analysis showed that the BrYV-C isolates formed a subgroup that was distinct from the BrYV-A and BrYV-B isolates based on all of the proteins except P5.

Biological and molecular variation of Iranian Cauliflower mosaic virus (CaMV) isolates.

Seventeen provinces of Iran were surveyed during 2003-2012 to find Brassicaceae hosts of Cauliflower mosaic virus (CaMV). A total 397 samples were collected from plants with virus-like symptoms. Among those tested by ELISA, 255 samples (67.2 %) were found to be infected with CaMV. Mechanical transmission tests showed that the Iranian isolates have similar biological properties on a number of Brassica and Raphanus plant species and cultivars tested. However, the isolates varied in the severity of symptoms they induced and in the capacity to infect B. oleracea var. capitata, on the basis of which they were grouped into two distinct biotypes L/MMo (latent/mild mottle) and severe (S) infection. The molecular diversity of natural population of CaMV were investigated based on the complete sequences of OFR 6 of 36 Iranian isolates collected from different geographically distant regions in Iran alongside the sequences of 14 previously reported isolates. Phylogenetic analyses indicated that the Iranian CaMV isolates belong to two groups (GI and GII). Most of the Iranian isolates fell into GI with other exotic isolates; however, the isolates from North-East Iran with Xinjiang from China fell into GII. The phylogenetic group GII (the North-East Iranian isolates) closely corresponded to the S biological group however other Iranian isolates corresponded to the L/MMo biological group. The within-population diversity was lower than the between population diversity suggesting the contribution of a founder effect on diversification of CaMV isolates. The Iranian isolates were differentiated from other exotic CaMV isolates and clustered into two RFLP groups using Hpy99I which closely corresponded to the biological and phylogenetic groups. This study showed the evolutionary process in CaMV isolates is shaped by a combination of host range differentiation and nucleotide substitution using the approach of population genetics.

Molecular characterization of a trisegmented chrysovirus isolated from the radish Raphanus sativus.

Radish (Raphanus sativus L.) is cultivated worldwide and is of agronomic importance. dsRNAs associated with partitiviruses were previously found in many R. sativus varieties. In this study, three large dsRNAs from radish were cloned using a modified single primer amplification technique. These three dsRNAs-of lengths 3638, 3517 and 3299 bp-shared conserved untranslated terminal regions, and each contained a major open reading frame putatively encoding the chrysoviral replicase, capsid protein and protease respectively. Isometric virus-like particles (VLP), approximately 45nm in diameter, were isolated from the infected radish plants. Northern blotting indicated that these dsRNAs were encapsidated in the VLP. The virus containing these dsRNA genome segments was named Raphanus sativus chrysovirus 1 (RasCV1). Phylogenetic analysis revealed that RasCV1 is a new species of the Chrysoviridae family and forms a plant taxon with another putative plant chrysovirus, Anthurium mosaic-associated virus (AmaCV). Furthermore, no fungal mycelia were observed in radish leaf tissues stained with trypan blue. These results indicated that RasCV1 is most likely a plant chrysovirus rather than a chrysovirus in symbiotic fungi. An exhaustive BLAST analysis of RasCV1 and AmaCV revealed that chrysovirus-like viruses might widely exist in eudicot and monocot plants and that endogenization of chrysovirus segments into plant genome might have ever happened.

Construction of an infectious cDNA clone of radish mosaic virus, a crucifer-infecting comovirus.

Radish mosaic virus (RaMV) is a crucifer-infecting comovirus that has been detected worldwide. Here, we report the successful construction of a full-length infectious cDNA clone of RaMV. The full-length cDNA clones corresponding to RNA1 and RNA2 of a Japanese isolate of RaMV were cloned into the pBlueScript plasmid or the binary vector pCAMBIA1301 downstream of the cauliflower mosaic virus 35S promoter. Mechanical inoculation or agroinoculation of Nicotiana benthamiana with these vectors resulted in systemic RaMV infections causing symptoms similar to those caused by the wild-type parental virus. The presence of progeny virus was verified by western blot analysis and electron microscopy.

Biology and interactions of two distinct monopartite begomoviruses and betasatellites associated with radish leaf curl disease in India.

BACKGROUND: Emerging whitefly transmitted begomoviruses are major pathogens of vegetable and fibre crops throughout the world, particularly in tropical and sub-tropical regions. Mutation, pseudorecombination and recombination are driving forces for the emergence and evolution of new crop-infecting begomoviruses. Leaf curl disease of field grown radish plants was noticed in Varanasi and Pataudi region of northern India. We have identified and characterized two distinct monopartite begomoviruses and associated beta satellite DNA causing leaf curl disease of radish (Raphanus sativus) in India. RESULTS: We demonstrate that RaLCD is caused by a complex of two Old World begomoviruses and their associated betasatellites. Radish leaf curl virus-Varanasi is identified as a new recombinant species, Radish leaf curl virus (RaLCV) sharing maximum nucleotide identity of 87.7% with Tomato leaf curl Bangladesh virus-[Bangladesh:2] (Accession number AF188481) while the virus causing radish leaf curl disease-Pataudi is an isolate of Croton yellow vein mosaic virus-[India] (CYVMV-IN) (Accession number AJ507777) sharing 95.8% nucleotide identity. Further, RDP analysis revealed that the RaLCV has a hybrid genome, a putative recombinant between Euphorbia leaf curl virus and Papaya leaf curl virus. Cloned DNA of either RaLCV or CYVMV induced mild leaf curl symptoms in radish plants. However, when these clones (RaLCV or CYVMV) were individually co-inoculated with their associated cloned DNA betasatellite, symptom severity and viral DNA levels were increased in radish plants and induced typical RaLCD symptoms. To further extend these studies, we carried out an investigation of the interaction of these radish-infecting begomoviruses and their associated satellite, with two tomato infecting begomoviruses (Tomato leaf curl Gujarat virus and Tomato leaf curl New Delhi virus). Both of the tomato-infecting begomoviruses showed a contrasting and differential interaction with DNA satellites, not only in the capacity to interact with these molecules but also in the modulation of symptom phenotypes by the satellites. CONCLUSION: This is the first report and experimental demonstration of Koch's postulate for begomoviruses associated with radish leaf curl disease. Further observations also provide direct evidence of lateral movement of weed infecting begomovirus in the cultivated crops and the present study also suggests that the exchange of betasatellites with other begomoviruses would create a new disease complex posing a serious threat to crop production.

Characterization of Phi2954, a newly isolated bacteriophage containing three dsRNA genomic segments.

BACKGROUND: Bacteriophage Phi12 is a member of the Cystoviridae and is distinct from Phi6, the first member of that family. We have recently isolated a number of related phages and five showed high similarity to Phi12 in the amino acid sequences of several proteins. Bacteriophage Phi2954 is a member of this group. RESULTS: Phi2954 was isolated from radish leaves and was found to have a genome of three segments of double-stranded RNA (dsRNA), placing it in the Cystoviridae. The base sequences for many of the genes and for the segment termini were similar but not identical to those of bacteriophage Phi12. However, the host specificity was for the type IV pili of Pseudomonas syringae HB10Y rather than for the rough LPS to which Phi12 attaches. Reverse genetics techniques enabled the production of infectious phage from cDNA copies of the genome. Phage were constructed with one, two or three genomic segments. Phage were also produced with altered transcriptional regulation. Although the pac sequences of Phi2954 show no similarity to those of Phi12, segment M of Phi2954 could be acquired by Phi12 resulting in a change of host specificity. CONCLUSIONS: We have isolated a new member of the bacteriophage family Cystoviridae and find that although it shows similarity to other members of the family, it has unique properties that help to elucidate viral strategies for genomic packaging and gene expression.

Evolutionary trajectory of turnip mosaic virus populations adapting to a new host.

Little is known about how some plant viruses establish successful cross-species transmission whilst others do not; the genetic basis for adaptation is largely unknown. This study investigated the genetic changes that occurred using the progeny of an infectious clone, p35Tunos, derived from the turnip mosaic virus (TuMV) UK 1 isolate, which has a Brassica host type, but rarely infects Raphanus systemically and then only asymptomatically. The genetic trajectory leading to viral adaptation was studied in a TuMV isolate passaged in Nicotiana benthamiana (parental), Brassica rapa, the old (susceptible) host and Raphanus sativus, the new (almost insusceptible) host. Almost-complete consensus genomic sequences were obtained by RT-PCR of viral populations passaged up to 35 times together with 59 full sequences of 578,200 nt. There were significant differences in the nucleotide and encoded amino acid changes in the consensus genomes from the old and new hosts. Furthermore, a 3264 nt region corresponding to nt 3222-6485 of the UK 1 genome was cloned, and 269 clones from 23 populations were sequenced; this region covered 33 % of the genome and represented a total of 878,016 nt. The results showed that the nucleotide diversity and the non-synonymous/synonymous ratio of the populations from the new host were higher than those from the old host. An analysis of molecular variance showed significant differences among the populations from the old and new hosts. As far as is known, this is the first report comparing the evolutionary trajectory dynamics of plant virus populations in old and new hosts.